ZIKV Virion Purification by Sucrose Gradient

The VLPs and ZIKV virions were purified by sucrose gradient ultracentrifugation as previously described [18 (link)]. Briefly, Sf9 cells infected with the recombinant baculovirus vAc-ZE or vAc-ZprME at a multiplicity of infection (MOI) of five. At three days post infection (d p.i.), culture supernatants were collected, cleared of cell debris and then concentrated using a 20% sucrose cushion at 150,000 g (SW41 rotor; Beckman, Brea, CA, USA) for 3 h. The pellets were resuspended in NTE buffer (120 mM NaCl, 10 mM Tris-HCl and 1 mM ethylenediaminetetraacetic acid [EDTA], pH 7.5) and applied to a continuous sucrose gradient (10–60%). Following ultracentrifugation at 150,000 g (SW41 rotor; Beckman) for 3 h, 12 fractions (from top to bottom) were collected for subsequent Western blot analysis and transmission electron microscopy (TEM) assays.